Novel bacteriophage lambda cloning vector.

Abstract

A simple method for generating phage collections representing eukaryotic genomes has been developed by using a novel bacteriophage A vector, X1059. The phage is a BamHI substitution vector that accommodates DNA fragments 6-24 kilobases long. Production of recombinants in X1059 requires deletion of the X red and gamma genes. The recombinants are therefore spiP and may be separated from the spi+ vector phages by plating on strains lysogenic for bacteriophage Pg. Random fragments suitable for insertion into X1059 are obtained by partial digestion of high molecular weight eukaryotic DNA with Sau3a. This restriction enzyme cleaves at the sequence G-A-T-C and leaves a 5'-tetranucleotide "sticky end." Because GA-T-C extensions are also produced by BamHI cleavage, these fragments may be annealed directly to BamHI-cleaved X1059. By using these methods, a set of clones covering the entire Caenorhabditis elegans genome was constructed. DNA segments which include the unc-54 myosin heavy chain gene have been isolated from this collection. Fractionation of genomes was an intractable problem until the introduction of recombinant DNA techniques. These methods eliminate the necessity for physical separations of DNA segments and permit the isolation of structural genes from collections of randomly cloned DNA fragments (1-10).Given a suitable probe, any eukaryotic gene may be isolated from a pool of cloned fragments, provided that it is large enough to give sequence representation of an entire genome. The simple multicellular eukaryote Caenorhabditis elegans has a haploid DNA content of approximately 8 X 107 base pairs (bp) (11). If random DNA cleavage and uniform cloning efficiency are assumed, a collection of 8 X 104 clones with an average length of 104 bp will be sufficient to include any genomic sequence with >99% probability. Similarly, the human genome with 2 X 109 bp will be covered by 106 clones 104 bp long.We have developed a novel bacteriophage X cloning vector, X1059, with properties that simplify the construction of recombinant phage collections representing genomes. The strategy is shown in Fig. 1 The structure of A1059 is shown in Fig. 2. The phage is a BamHI substitution vector composed of three BamHI fragments: a 19.6-kb left arm carrying the genes for the A head and tail proteins, a 17-kb central fragment, and a 9.4-kb right arm carrying the A replication and lysis genes. The two arms of the vector contain all the essential functions required for A replication and maturation in a DNA sequence 58.2% of the wildtype length. Viable phages are produced when these arms are hybridized with internal DNA fragments 12.8% to 49.8% the length of wild-type A (6.3-24.4 kb). The two arms alone do not produce viable phages, because lambdoid phages require genome sizes between 70% and 108% of the wild-type DNA to fill the phage heads properly (37, 38).The central BamHI fragment of the vector balancing the phage arms carried the A red (exo and 3 genes) and gamma functions under the control of the leftward promoter (pL) an...

Variation at the 87A heat shock locus in Drosophila melanogaster

“…The ligated mix was packaged in vitro following Scalenghe et al (37) and screened as above, omitting the chloramphenicol amplification step. The 87A hsp70 loci from lines NC903 and NC960 were cloned in AEMBL4, a derivative of 1059 (38) Genetics: Leigh Brown The heterozygosity due to these insertions is estimated to be 0.67. A total of 13 kb of DNA, of which 9 kb was noncoding, was screened in sufficient detail to detect events of this size.…”

Cloning, sequencing and expression of the gene encoding the carboxytransferase subunit of the biotin‐dependent Na+ pump glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans in Escherichia coli

“…The genomic library of A. fernentuns was constructed with the 2 vector EMBL12 (Natt and Scherer, 1986) and amplified in E. coli Q358 (Karn et al, 1980). This strain was grown aerobically on Luria-Bertoni medium (Sambrook et al, 1989).…”

Identification by sequence analysis of two-site posttranslational processing of the cysteine-rich outer membrane protein 2 of Chlamydia trachomatis serovar L2

“…C. trachomatis L2/434/Bu, B/TW5/OT, C/TW3/OT, F/UW6/Cx and I/UW-12/Ur have been previously described (15), as has the Xgt11 bacteriophage recombinant from serovar L2 designated gtll/L2/46 (24). Bacteriophage X1059, its host Escherichia coli Q359 (12), and the pUC plasmids and M13 phage systems, have been previously described (18).…”

“…The Saq3A fragments were ligated to BamHI-digested X1059 DNA and packaged into phage by using commercially prepared extracts (Amersham Corp.). Recombinant phage were plated on Q359 for screening (12). Selection and analysis of X1059 recombinant phage.…”